RCE­4, a potential anti­cervical cancer drug isolated from Reineckia carnea, induces autophagy via the dual blockade of PI3K and ERK pathways in cervical cancer CaSki cells.

The steroidal saponin RCE­4 (1ß, 3ß, 5ß, 25S)­spirostan­1, 3­diol 1­[α­L­rhamnopyranosyl­(1→2)­ß­D­xylopyranoside], isolated from Reineckia carnea, exerts significant anti­cervical cancer activity by inducing apoptosis. The potential effect of RCE­4 on proliferation inhibition and autophagy induction has rarely been studied. Therefore, the focus of the present study was to investigate the effects of RCE­4 on proliferation, and to elucidate the detailed mechanisms involved in autophagy induction in cervical cancer cells. CaSki cells were treated with RCE­4 or/and autophagy inhibitors, and the effect of RCE­4 on cellular proliferation was assessed by MTT assay. The pro­autophagic properties of RCE­4 were subsequently confirmed using monomeric red fluorescent protein­green fluorescent protein­microtubule­associated proteins 1A/1B light chain 3B (LC3) adenoviruses and CYTO­ID autophagy assays, and by assessing the accumulation of lipid­modified LC3 (LC3II). The mechanisms of RCE­4­induced autophagy were investigated by western blot analysis. The results demonstrated that inhibiting autophagy significantly promoted RCE­4­induced cell death, indicating that autophagy served a protective role following RCE­4 treatment. In addition, RCE­4­induced autophagy was reflected by increased expression levels of the serine/threonine­protein kinase ULK1, phosphorylated (p)­ULK1, p­Beclin­1 and LC3II, the formation of autophagosomes and autolysosomes, and sequestosome 1 (p62) degradation. Subsequent analysis indicated that RCE­4 activated the AMP­activated protein kinase (AMPK) pathway by upregulating AMPK and p­AMPK, and also inhibited the PI3K and extracellular signal­regulated kinase (ERK) signaling pathways by downregulating p­PI3K, p­Akt, p­mTOR, Ras, c­Raf, p­c­Raf, dual specificity mitogen­activated protein kinase kinase (MEK)1/2, p­MEK1/2 and p­Erk1/2. Additionally, with increased treatment times RCE­4 may impair lysosomal cathepsin activity and inhibit autophagy flux by suppressing the expression of AMPK, p­AMPK, ULK1, p­ULK1 and p­Beclin­1, and upregulating that of p62. These results indicated that the dual RCE­4­induced inhibition of the PI3K and ERK pathways may result in a more significant anti­tumor effect and prevent chemoresistance, compared with the inhibition of either single pathway; furthermore, dual blockade of PI3K and ERK, and the AMPK pathway may be involved in the regulation of autophagy caused by RCE­4. Taken together, RCE­4 induced autophagy to protect cancer cells against apoptosis, but AMPK­mediated autophagy was inhibited in the later stages of RCE­4 treatment. In addition, autophagy inhibition improved the therapeutic effect of RCE­4. These data highlight RCE­4 as a potential candidate for cervical cancer treatment.

Polarograhic adsorptive wave of protein hydrolysate in Pb2+ and sodium hydroxide solution and its application / 药学学报

<p><b>AIM</b>To propose a new simple and sensitive voltammetric method for determination of proteins.</p><p><b>METHODS</b>Protein with sulfhydryl or disulfide bond in 0.5 mol x L(-1) NaOH, 1.5 x 10(-4) mol x L(-1) Pb2+ and 0.02% tetrabutylammonium iodide was heated in boiling water for 5 minutes. The reactive product gave a well defined reductive adsorption wave at -0.66 V (vs SCE) by means of single sweep polarography, and the height of derivative wave was proportional to the concentration of proteins.</p><p><b>RESULTS</b>The peak height was linearly proportional to bovine serum albumin (BSA) or human serum albumin (HSA) concentration in range of 7.5 x 10(-10) -3.0 x 10(-7) mol x L(-1) (r(BSA) = 0.9995, and r(HSA) = 0.9990). The detection limit of BSA or HSA was 3.0 x 10(-10) mol x L(-1). For lysozyme (Lyso), the concentration range was from 1.4 x 10(-8) to 1.3 x 10(-6) mol x L(-10 (r(Lyso) = 0.9997) and the detection limit was 7.0 x 10(-9) mol x L(-1).</p><p><b>CONCLUSION</b>The method is simple, rapid, sensitive and applicable to the assay of diluted human serum albumin samples.</p>